This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Keratinocytes in vivo undergo an epithelial to mesenchymal transition (EMT) during carcinogenesis. This phenotype is characterized by loss of e-cadherin and zo-1 staining in junctional complexes, and an increase in vimentin staining and vinculin-rich focal adhesions. Cells become solitary, migratory and invasive. Increased activity of matrix metalloproteinases (MMPs) is correlated with EMT. While genetic changes have been ascribed to EMT, the environment is in part responsible for the EMT phenotype, due to the observation in vivo that carcinogenic keratinocytes undergo the reverse mesenchymal to epithelial transition (MET) after migrating into other tissue sites. We previously showed that ectopic H-ras expression in keratinocytes was sufficient for their invasion into the dermal compartment of a skin equivalent. Correlated with increase in basement membrane protein deposition, invasion stopped after 3 weeks. We hypothesized that the environmental change was sufficient to inhibit invasion. The purpose of this proposal is to study the environment's contribution to keratinocyte invasion and correlated EMT, as well as the reverse MET phenotype using three different variables in a skin equivalent model. Ras-overexpressing keratinocytes will be plated onto de-epidermized dermis (DED) with a pre-formed basement membrane to inhibit invasion as the first Specific Aim. Preliminary studies indicate that this model inhibits invasion and EMT. A second specific aim will determine whether TGF-beta1, a known inducer of EMT, will override the inhibitory effects of the DED model. A third specific aim will determine whether fibroblasts incorporated in the skin equivalent will be sufficient to induce EMT in the DED model. Analysis of each aim will include immunofluorescence staining to determine the changes in junctional complexes and cytoskeleton characteristic of each phenotype;PCR and Western Blotting to monitor transcription and expression changes of MMPs. Each specific aim will be lead by one upperclassman with the assistance of at least one underclassman, with the ultimate goal of disseminating results through presentations and a manuscript.